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At least 199 records · Page 11

Research for amino acids in lunar samples.

The study was primarily directed toward the examination of Apollo 14 lunar fines for indigenous amino acids or materials which could be converted to amino acids on hydrolysis with 6 N hydrochloric acid. Initial experiments were conducted to confirm the integrity of the derivatization reactions and reagents, and to optimize the gas-liquid chromatographic (GLC) instrumental and chromatographic system for the separation and flame ionization detection of the amino acid derivatives. In studies on the recovery of amino acids added to lunar fines, low recoveries were obtained when 10 ng of each amino acid were added to 50 mg of virgin fines, but the subsequent addition of 50 ng of each to the previously extracted sample resulted in much higher recoveries.

Gehrke, C. W.↗

Parent Body Influences on Amino Acids in the Tagish Lake Meteorite

The Tagish Lake meteorite is a primitive C2 carbonaceous chondrite with a mineralogy, oxygen isotope, and bulk chemical. However, in contrast to many CI and CM carbonaceous chondrites, the Tagish Lake meteorite was reported to have only trace levels of indigenous amino acids, with evidence for terrestrial L-amino acid contamination from the Tagish Lake meltwater. The lack of indigenous amino acids in Tagish Lake suggested that they were either destroyed during parent body alteration processes and/or the Tagish Lake meteorite originated on a chemically distinct parent body from CI and CM meteorites where formation of amino acids was less favorable. We recently measured the amino acid composition of three different lithologies (11h, 5b, and 11i) of pristine Tagish Lake meteorite fragments that represent a range of progressive aqueous alteration in order 11h < 5b < 11i as inferred from the mineralogy, petrology, bulk isotopes, and insoluble organic matter structure. The distribution and enantiomeric abundances of the one- to six-carbon aliphatic amino acids found in hot-water extracts of the Tagish Lake fragments were determined by ultra performance liquid chromatography fluorescence detection and time of flight mass spectrometry coupled with OPA/NAC derivatization. Stable carbon isotope analyses of the most abundant amino acids in 11h were measured with gas chromatography coupled with quadrupole mass spectrometry and isotope ratio mass spectrometry.

Glavin, D. P.↗

Methods of making proteins with non-standard amino acids

The disclosure provides methods of making a protein having a desired non-standard amino acid incorporated at its N-terminus in a cell and methods of screening for an amino acyl tRNA synthetase variant that preferentially selects a non-standard amino acid against its standard amino acid counterpart or undesired non-standard amino acids for incorporation into a protein in a cell.

Kunjapur, Aditya Mohan↗

The Effects of Parent Body Processes on Amino Acids in Carbonaceous Chondrites

To investigate the effect of parent body processes on the abundance, distribution, and enantiomeric composition of amino acids in carbonaceous chondrites, the water extracts from nine different powdered Cl, CM, and CR carbonaceous chondrites were analyzed for amino acids by ultrahigh performance liquid chromatography-fluorescence detection and time-of-flight mass spectrometry (UPLC-FD/ToF-MS). Four aqueously altered type 1 carbonaceous chondrites including Orgueil (C11), Meteorite Hills (MET) 01070 (CM1), Scott Glacier (SCO) 06043 (CM1), and Grosvenor Mountains (GRO) 95577 (CR1) were analyzed using this technique for the first time. Analyses of these meteorites revealed low levels of two- to five-carbon acyclic amino alkanoic acids with concentrations ranging from -1 to 2,700 parts-per-billion (ppb). The type 1 carbonaceous chondrites have a distinct distribution of the five-carbon (C5) amino acids with much higher relative abundances of the gamma- and delta-amino acids compared to the type 2 and type 3 carbonaceous chondrites, which are dominated by a-amino acids. Much higher amino acid abundances were found in the CM2 chondrites Murchison, Lonewolf Nunataks (LON) 94102, and Lewis Cliffs (LEW) 90500, the CR2 Elephant Moraine (EET) 92042, and the CR3 Queen Alexandra Range (QUE) 99177. For example, a-aminoisobutyric acid ((alpha-AIB) and isovaline were approximately 100 to 1000 times more abundant in the type 2 and 3 chondrites compared to the more aqueously altered type 1 chondrites. Most of the chiral amino acids identified in these meteorites were racemic, indicating an extraterrestrial abiotic origin. However, non-racemic isovaline was observed in the aqueously altered carbonaceous chondrites Murchison, Orgueil, SCO 06043, and GRO 95577 with L-isovaline excesses ranging from approximately 11 to 19%, whereas the most pristine, unaltered carbonaceous chondrites analyzed in this study had no detectable L-isovaline excesses. These results are consistent with the theory that aqueous alteration played an important role in amplification of small initial left handed isovaline excesses on the parent bodies.

Glavin, Daniel P.↗

Water driven adsorption of amino acids on the (101) anatase TiO 2 surface: an ab initio study

Arg, Lys and Asp amino acids are known to play a critical role in the adhesion of the RKLPDA engineered peptide on the (101) surface of the titania anatase phase. In order to understand their contribution to peptide adhesion, we have considered the relevant charge states due to protonation (Arg and Lys) or deprotonation (Asp) occurring in neutral water solution, and studied their adsorption on the (101) anatase TiO 2 surface by ab initio total energy calculations based on density functional theory. The adsorption configurations on the hydrated surface are compared to those on the dry surface considering also the presence of the hydration shell around amino acid side-chains. Our study explains how water molecules mediate the adsorption of charged amino acids showing that protonated amino acids are chemically adsorbed much more strongly than de-protonated Asp. Finally, it is shown that the polar screening of the hydration shell reduces the adsorption energy of the protonated amino acids to a small extent, thus evidencing that both Arg and Lys strongly adhere on the (101) anatase TiO 2 surface in neutral water solution and that they play a major role in the adhesion of the RKLPDA peptide.

36 MATERIALS SCIENCE↗

Amino Acid Changes in Disease-Associated Variants Differ Radically from Variants Observed in the 1000 Genomes Project Dataset

The 1000 Genomes Project data provides a natural background dataset for amino acid germline mutations in humans. Since the direction of mutation is known, the amino acid exchange matrix generated from the observed nucleotide variants is asymmetric and the mutabilities of the different amino acids are very different. These differences predominantly reflect preferences for nucleotide mutations in the DNA (especially the high mutation rate of the CpG dinucleotide, which makes arginine mutability very much higher than other amino acids) rather than selection imposed by protein structure constraints, although there is evidence for the latter as well. The variants occur predominantly on the surface of proteins (82%), with a slight preference for sites which are more exposed and less well conserved than random. Mutations to functional residues occur about half as often as expected by chance. The disease-associated amino acid variant distributions in OMIM are radically different from those expected on the basis of the 1000 Genomes dataset. The disease-associated variants preferentially occur in more conserved sites, compared to 1000 Genomes mutations. Many of the amino acid exchange profiles appear to exhibit an anti-correlation, with common exchanges in one dataset being rare in the other. Disease-associated variants exhibit more extreme differences in amino acid size and hydrophobicity. More modelling of the mutational processes at the nucleotide level is needed, but these observations should contribute to an improved prediction of the effects of specific variants in humans.

59 BASIC BIOLOGICAL SCIENCES↗

Effects of amino acid substitutions on the biological activity of anti-CD20 monoclonal antibody produced by transgenic silkworms (Bombyx mori)

Highlights: • Amino acid substitutions modulate Fc-mediated functions of glyco-engineered mAbs. • Fc-engineered mAbs produced by silkworms show unique Fc-mediated functions. • Transgenic silkworm is a promising system for the production of Fc-engineered mAbs. Recombinant monoclonal antibodies (mAbs) have been used in various therapeutic applications including cancer therapy. Fc-mediated effector functions play a pivotal role in the tumor-killing activities of some tumor-targeting mAbs, and Fc-engineering technologies with glyco-engineering or amino acid substitutions at the antibody Fc region have been used to enhance cytotoxic activities including antibody-dependent cellular cytotoxicity (ADCC). We previously reported that the mAbs produced using transgenic silkworms showed stronger ADCC activity and lower complement-dependent cytotoxicity (CDC) activity than mAbs derived from Chinese hamster ovary (CHO) cells due to their unique N-glycan structure (lack of core-fucose and non-reducing terminal galactose). In this study, we generated anti-CD20 mAbs with amino acid substitutions using transgenic silkworms and analyzed their biological activities to assess the effect of the combination of glyco-engineering and amino acid substitutions on the Fc-mediated function of mAbs. Three types of amino acid substitutions at the Fc region (G236A/S239D/I332E, L234A/L235A, and K326W/E333S) modified the Fc-mediated biological activities of silkworm-derived mAbs as in the case of CHO-derived mAbs, resulting in the generation of Fc-engineered mAbs with characteristic Fc-mediated functions. The combination of amino acid substitutions at the Fc region and glyco-engineering using transgenic silkworm made it possible to generate Fc-engineered mAbs with suitable Fc-mediated biological functions depending on the pharmacological mechanism of their actions. Transgenic silkworms were shown to be a promising system for the production of Fc-engineered mAbs.

60 APPLIED LIFE SCIENCES↗

Enhanced Synthesis of Alkyl Amino Acids in Miller's 1958 H2S Experiment

Stanley Miller's 1958 H2S-containing experiment, which included a simulated prebiotic atmosphere of methane (CH4), ammonia (NH3), carbon dioxide (CO2), and hydrogen sulfide (H2S) produced several alkyl amino acids, including the alpha-, beta-, and gamma-isomers of aminobutyric acid (ABA) in greater relative yields than had previously been reported from his spark discharge experiments. In the presence of H2S, aspariic and glutamic acids could yield alkyl amino acids via the formation of thioimide intermediates. Radical chemistry initiated by passing H2S through a spark discharge could have also enhanced alkyl amino acid synthesis by generating alkyl radicals that can help form the aldehyde and ketone precursors to these amino acids. We propose mechanisms that may have influenced the synthesis of certain amino acids in localized environments rich in H2S and lightning discharges, similar to conditions near volcanic systems on the early Earth, thus contributing to the prebiotic chemical inventory of the primordial Earth.

Parker, Eric T.↗

Amino acid analysis

The process and apparatus for qualitative and quantitative analysis of the amino acid content of a biological sample are presented. The sample is deposited on a cation exchange resin and then is washed with suitable solvents. The amino acids and various cations and organic material with a basic function remain on the resin. The resin is eluted with an acid eluant, and the eluate containing the amino acids is transferred to a reaction vessel where the eluant is removed. Final analysis of the purified acylated amino acid esters is accomplished by gas-liquid chromatographic techniques.

Winitz, M.↗

Amino Acid Chemistry as a Link Between Small Solar System Bodies and Carbonaceous Chondrites

Establishing chemical links between meteorites and small solar system bodies, such as comets and asteroids, provides a tool for investigating the processes that occurred during the formation of the solar system. Carbonaceous meteorites are of particular interest, since they may have seeded the early Earth with a variety of prebiotic organic compounds including amino acids, purines and pyrimidines, which are thought to be necessary for the origin of life. Here we report the results of high-performance liquid chromatography (HPLC) based amino acid analyses of the acid-hydrolyzed hot water extracts from pristine interior pieces of the CI carbonaceous chondrites Orgueil and Ivuna and the CM meteorites Murchison and Murray. We found that the CI meteorites Orgueil and Ivuna contained high abundances of beta-alanine and glycine, while only traces of other amino acids like alanine, alpha-amino-n-butryic acid (ABA) and alpha-aminoisobutyric acid (AIB) were detected in these meteorites. Carbon isotopic measurements of beta-alanine and glycine in Orgueil by gas chromatography combustion-isotope ratio mass spectrometry clearly indicate an extraterrestrial origin of these amino acids. The amino acid composition of Orgueil and Ivuna was strikingly different from the CM chondrites Murchison and Murray. The most notable difference was the high relative abundance of B-alanine in Orgueil and Ivuna compared to Murchison and Murray. Furthermore, AIB, which is one of the most abundant amino acids found in Murchison and Murray, was present in only trace amounts in Orgueil and Ivuna. Our amino acid data strongly suggest that the CI meteorites Orgueil and Ivuna came from a different type of parent body than the CM meteorites Murchison and Murray, possibly from an extinct comet. It is generally thought that carbonaceous meteorites are fragments of larger asteroidal bodies delivered via near Earth objects (NEO). Orbital and dynamic studies suggest that both fragments of main belt asteroids and comets replenish the NEO population, therefore extinct comets may contribute up to half of all NEO's. A comparison of an amino acid analysis of a returned NEO sample to CI and CM carbonaceous chondrites would help establish a link between small solar system bodies and meteorites. Based on our amino acid measurements of CI and CM chondrites, amino acid chemistry can be included as an additional set of criteria to constrain the nature of meteorite parent bodies.

Glavin, Daniel P.↗

Factors influencing the rate of non-enzymatic activation of carboxylic and amino acids by ATP

The nonenzymatic formation of adenylate anhydrides of carboxylic and amino acids is discussed as a necessary step in the origin of the genetic code and protein biosynthesis. Results of studies are presented which have shown the rate of activation to depend on the pKa of the carboxyl group, the pH of the medium, temperature, the divalent metal ion catalyst, salt concentration, and the nature of the amino acid. In particular, it was found that of the various amino acids investigated, phenylalanine had the greatest affinity for the adenine derivatives adenosine and ATP. Results thus indicate that selective affinities between amino acids and nucleotides were important during prebiotic chemical evolution, and may have played a major role in the origin of protein synthesis and genetic coding.

Mullins, D. W., Jr.↗

Production of carrier-peptide conjugates using chemically reactive unnatural amino acids

Provided are methods of making carrier polypeptide that include incorporating a first unnatural amino acid into a carrier polypeptide variant, incorporating a second unnatural amino acid into a target polypeptide variant, and reacting the first and second unnatural amino acids to produce the conjugate. Conjugates produced using the provided methods are also provided. In addition, orthogonal translation systems in methylotrophic yeast and methods of using these systems to produce carrier and target polypeptide variants comprising unnatural amino acids are provided.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Production of carrier-peptide conjugates using chemically reactive unnatural amino acids

Provided are methods of making carrier polypeptide that include incorporating a first unnatural amino acid into a carrier polypeptide variant, incorporating a second unnatural amino acid into a target polypeptide variant, and reacting the first and second unnatural amino acids to produce the conjugate. Conjugates produced using the provided methods are also provided. In addition, orthogonal translation systems in methylotrophic yeast and methods of using these systems to produce carrier and target polypeptide variants comprising unnatural amino acids are provided.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Production of carrier-peptide conjugates using chemically reactive unnatural amino acids

Provided are methods of making carrier polypeptide that include incorporating a first unnatural amino acid into a carrier polypeptide variant, incorporating a second unnatural amino acid into a target polypeptide variant, and reacting the first and second unnatural amino acids to produce the conjugate. Conjugates produced using the provided methods are also provided. In addition, orthogonal translation systems in methylotrophic yeast and methods of using these systems to produce carrier and target polypeptide variants comprising unnatural amino acids are provided.

59 BASIC BIOLOGICAL SCIENCES↗